Article pubs.acs.org/JAFC

Inhibitory Effect of Liposomal Solutions of Grape Seed Extract on the Formation of Heterocyclic Aromatic Amines Daniela Natale,† Monika Gibis,*,‡ Maria Teresa Rodriguez-Estrada,† and Jochen Weiss‡ †

Department of Agricultural and Food Sciences, Alma Mater Studiorum-Università di Bologna, Viale Fanin 40, 40127 Bologna, Italy Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany

‡

S Supporting Information *

ABSTRACT: The effectiveness of grape seed extract (GSE) encapsulated in liposomes to inhibit the formation of heterocyclic aromatic amines (HAA) during frying of beef patties was assessed. All liposomal systems were prepared by high pressure homogenization at 22 500 psi. A total of six samples (rapeseed oil (control), GSE at 0.1% and 0.2%, and GSE-containing liposomes with 1%, 2%, and 5% soy lecithin) were investigated. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f ]quinoxaline), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine), Norharman, and Harman were found after the marinade application and frying. PhIP concentrations decreased upon marination with GSE (0.1%) and GSE-containing liposomes (1% and 5%) (p < 0.05). MeIQx contents decreased in all samples compared to the oil control (p < 0.01) while no effect on β-carboline formation was observed. Results are in contrast to previous studies that had shown that liposomal encapsulation may enhance effectiveness of polyphenols to inhibit radical reactions. A mechanistic model was proposed to explain the observed differences. KEYWORDS: liposomes, grape seed extract, heterocyclic aromatic amines, phenolic compounds, beef patties

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INTRODUCTION Diet plays a vital role in maintaining human health, and many studies have been carried out that demonstrate a cause and effect relationship between diet and disease, especially if harmful substances are present in food. Heterocyclic aromatic amines (HAA) are such compounds, as they have been shown to exhibit carcinogenic and mutagenic activities. They are formed in meat and fish when cooked at high temperatures (≥150 °C).1,2 HAA are classified into two groups based on the temperatures that are prevalent during their formation. HAA formed at temperatures between 100 °C and 300 °C are known as “thermic HAA”, IQ type, or aminoimidazoazaarenes (AIA) while HAA produced at higher temperatures (>300 °C) are known as “pyrolytic HAA” or non-IQ type.3 Their concentration has been found to depend on meat type, cooking parameters (duration, temperature, equipment, and methods), pH, water activity, creatine concentration, amount, and type of carbohydrates, as well as free amino acid content.4 Several studies have demonstrated the progressive decrease of HAA in the presence of compounds with antioxidant activity, such as vitamin E, fruit extracts, plant extracts, carotenoids, spices, beer, wine, and polyphenols.5−8 A wide variety of polyphenols exist in nature, with compounds exhibiting significant antioxidant activities thereby inhibiting radical reactions. Moreover, they have been found to display beneficial physiological functions, such as, for example, anticarcinogenic activity.9 These potent antioxidants, however, can lose their efficacy when subjected to high temperatures or exposed to oxygen or light-sources. To improve their stability and also control release, researchers have worked on a rational design of encapsulation systems that are compatible with the application matrix and enable protection from degradation.10,11 Such encapsulation approaches have shown to not only © XXXX American Chemical Society

improve chemical stability but also to alleviate unpleasant taste or flavor and improve the compounds’ in vivo and in vitro bioavailabilities.12 Different studies have demonstrated that the activity of polyphenols in particular may be maintained and/or enhanced by encapsulation.13 Microencapsulation can be performed by different techniques, such as spray drying, spray chilling, fluidized bed coating, extrusion coating, coacervation, inclusion complexation, centrifugal extrusion, rotational suspension separation, and liposome entrapment.14 The latter method involves the use of lipid bilayer vesicles that enclose aqueous compartments, allowing in principal the conveyance of a wide variety of molecules having different polarities. Recent studies have shown that liposomes are particularly well suited to carry bioactive compounds of an amphiphilic nature.15 Liposomal encapsulation, therefore, is a powerful technological tool that can be helpful in producing high-quality foods. The controlled delivery of bioactive compounds can lead to an enhanced absorption of such molecules and to food systems that are more stable having extended shelf-lives.16−18 The polyphenols in grape seed extract (GSE) consist mainly of flavonoids, including gallic acid, monomeric flavan-3-ols catechin, epicatechin, gallocatechin, epigallocatechin, and epicatechin 3-O-gallate, and procyanidin dimers and trimers and the more highly polymerized procyanidin. 19 Such compounds, when encapsulated in liposomes, have shown to be predominately localized in the bilayer membrane, and only a small part of the substances are found in the inner Received: April 16, 2013 Revised: December 8, 2013 Accepted: December 9, 2013

A

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Table 1. Experimental Design Used for This Study (each treatment, n = 6)a systems controls

(1) no liposomes (0% of soy lecithin)

liposomes

(1) liposome with 1% of soy lecithin

treatments (a) patties marinated with rapeseed oil (b) patties marinated with pure GSE (0.1%) (c) patties marinated with pure GSE (0.2%) (a) control without GSE (b) marinated with 0.1% of GSE (c) marinated with 0.2% of GSE (a) control without GSE (b) marinated with 0.1% of GSE (c) marinated with 0.2% of GSE (a) control without GSE (b) marinated with 0.1% of GSE (c) marinated with 0.2% of GSE

(2) liposome with 2% of soy lecithin (3) liposome with 5% of soy lecithin a

Seventy-two deep-frozen beef patties marinated with 10 g of each solution.

compartment of the liposomes.20 In model systems these capsules have proven to be highly effective in inhibiting radical reactions.21 However, only a few studies have been conducted that have investigated their functionalities in complex foods such as, for example, meats and meat products. The aim of this work was to evaluate the efficiency of the encapsulation of polyphenols from GSE in liposomes and to assess their functionality with respect to HAA formation in fried beef patties after marination. We hypothesized that liposomalentrapped GSE may have a higher inhibitory effect on the formation of HAA, as liposomes provide large surfaces which can readily interact with the meat compounds that generate HAA. Moreover, liposomes could facilitate a burst release upon heating, which might be beneficial to inhibit the initial stages of the propagating reaction.

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was dispersed in an acetate buffer system (0.25 mol/L, pH 3.8) containing GSE at 0.1% and 0.2%. The GSE-containing buffer system was prepared in ultrapure water and filtered with a folded cellulose filter (Whatman-VWR, Darmstadt, Germany). The GSE solution had been characterized in an earlier study by LC/MS/MS,23 by a method suggested by Maier et al.19 The main components of the filtered extract were procyanidin B1 (19.3 mg/g), (+)-catechin (19.1 mg/g), (−)-epicatechin (10.3 mg/g), procyanidin B2 (5.3 mg/g), gallic acid (2.6 mg/g), and (−)-epicatechin gallate (2.1 mg/g). Galloylated dimeric procyanidin and dimeric procyanidins were also found by HPLC-ESI-MS/MS.19 The mixture was predispersed using a magnetic stirrer overnight and was passed five times through a high-pressure homogenizer consisting of a diamond chamber at 22 500 psi (M110EH-30, Microfluidics International Cooperation, Newton, MA) to form finely dispersed liposomes incorporating GSE. Zeta Potential and Average Particle Diameter Measurements of Liposomes. Liposomal charge and z-average particle diameter were determined by dynamic light-scattering (Zetasizer Nano ZS, model ZEN3600, Malvern Instruments, Malvern, UK) at 25 °C. The samples were diluted approximately nine times with the acetate buffer (0.25 mol/L, pH 3.8) and transferred into cuvettes for size determination (DTS1060, Malvern Instruments). The instrument measures the size of particles by determining their Brownian motion at a backscattering angle of 173°. The charge of particles is determined by applying an oscillating electric field and measuring the electrophoretic mobility of particles via light scattering. The z-average particle size and zeta potential were determined immediately after liposome preparation and throughout the four weeks of storage. For each sample, the measurement was repeated twice at 25 °C. Determination of Total Phenolic Compounds by Folin− Ciocalteu Assay. Two GSE solutions (0.1% and 0.2%) were prepared by dissolving the dried GSE in acetate buffer (pH 3.8 ± 0.2; 0.25 M), stirring for 30 min, and filtering with folded filters. The amount of phenolic compounds was determined in the extract and in the liposomes after encapsulation and after gel filtration according to the Folin−Ciocalteu assay.24 Liposomes were deliberately destabilized by adding 3 mL of 15 wt % Triton X100 to the samples for the determination of polyphenols in bilayers. The spectrophotometric measurements were carried out at 720 nm, after a 60-min reaction. The results were expressed as milligrams of gallic acid per gram of extract. The encapsulation efficiency of liposomes containing extract was calculated by the amount of total polyphenols of liposomes containing GSE after gel filtration and treatment with the surfactant Triton X-100 divided by the concentration of total polyphenols treated with Triton X-100. Preparation of Samples and Pan-Frying Trials. Deep-frozen beef patties were obtained from Salomon Hitburger (Grossostheim, Germany). A total of 72 deep-frozen beef patties (62.5 g, 112 × 105 mm, thickness 10 mm) were marinated with 10 g of marinade solution (n = 6 for each experimental treatment). As controls, a treatment marinated with rapeseed oil and two treatments marinated with 0.1% and 0.2% unencapsulated (free) GSE were used. Liposomal treatments consisted of solutions containing 1%, 2%, and 5% of soy lecithin and two different GSE concentrations (0.1% and 0.2%) (Table 1). The

MATERIALS AND METHODS

Materials. Soy lecithin (Lipoid S75) was provided by Lipoid (Ludwigshafen, Germany). The contents of phosphatidylcholine, phosphatidylethanolamine, and lysophosphatidylcholine were 69.3%, 9.8%, and 2.1%, respectively, according to the company’s specifications. The concentration of the antioxidant DL-α-tocopherol was 0.18%, and the fresh lecithin standard had a maximal peroxide value specification of 3 mequiv O2/kg (1.5 mmol O2/kg). The grape seed extract (GSE; Vitis vinifera L.) had a procyanidins content of ≥30% (calculated as cyanidin chloride) and a total content of polyphenols of ≥40% (calculated as anhydrous gallic acid) according to the manufacturer’s specification; GSE was provided by the Martin Bauer Group Plantextrakt GmbH & Co.KG. (Vestenbergsgreuth, Germany). SephadexG50, Triton X100, gallic acid, sodium acetate, acetic acid, hydrochloric acid, ammonium hydroxide solution, sodium hydroxide, and Folin−Ciocalteu reagent were purchased from Sigma-Aldrich (St. Louis, MO). Ethyl acetate, toluene, methanol, acetonitrile (HPLC grade), triethylamine, and orthophosphoric acid (20%) were supplied by Merck (Darmstadt, Germany). A silica column, propylsulfonic acid (PRS) (500 mg), and C18 (500 mg and 100 mg) solid-phase extraction (SPE) cartridges were purchased from Varian (Palo Alto, CA). The HAA standards IQ, IQx, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8DiMeIQx, PhIP, Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2, AαC, and MeAαC were obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Norharman and harman were supplied by SigmaAldrich (St. Louis, MO). The standards were used to prepare a mixed stock standard solution with final concentration of 19.7, 22.8, 21.3, 13.6, 12.9, 12.5, 9.6, 6.5, 5.9, 7.4, 9.4, 5.3, 5.0, 4.9, and 4.1 ng in 100 μL of methanol, respectively (concentrations of HAA in order as described above). All stock solutions were corrected by means of the extinction coefficient.22 Caffeine was used as an internal standard at a concentration of 2.5 μg/mL in ultrapure water and methanol (1/1, v/v). Preparation of Liposomes. Liposomes were prepared with different percentages of soy lecithin (1%, 2%, and 5%). Soy lecithin B

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marination time was 3 h at 8 °C. The marinated beef patties were fried on a double contact grill (Nevada, Neumarker, Hemer, Germany) and heated at 220 °C for 2 min 40 s, as previously described by Gibis et.6 The core temperature was monitored using a data logger (Therm 3280-8M, Ahlborn, Holzkirchen, Germany). Meat samples were stored under vacuum conditions in the freezer (−18 °C) until analysis. All measurements were repeated at least three times using two replicates. Means and standard deviations were calculated from these measurements with Excel (Microsoft, Redmond, VA). Determination of HAA. Concentrations of 15 polar and nonpolar HAA were determined using a modified HPLC method25 based on the method described by Gross and Grueter.26 Briefly, crushed patties were homogenized with 1 M sodium hydroxide solution. Two homogenates were spiked with 100 μL of the standard mixture. Diatomaceous earth was added and mixed to each of the four equivalent homogenates. HAA were extracted from the patties by multiple solid-phase extractions. HAA were eluted with an ethyl acetate solution containing 5% toluene (100 mL) and adsorbed onto a coupled preconditioned cation exchanger PRS cartridge which had been preconditioned with a mixture of ethyl acetate and 5% toluene. After the PRS cartridge had been dried and washed with 0.01 M HCl solution, the nonpolar HAA were eluted with 15 mL of a mixture of 0.01 M HCl:methanol (2:3, v/v). The nonpolar fraction was purified, first by being adsorbed onto a C18 cartridge (500 mg). After a cleaning step with ultrapure water and drying, the nonpolar HAA were eluted with a 1.2 mL of methanol:ammonia (25%) mixture (90:10, v/ v) into a vial. After drying, the eluate was redissolved in 100 μL of caffeine solution (internal standard). The polar HAA were eluted from the PRS cartridges with 20 mL of 0.5 M ammonia acetate solution (pH 8.5) and adsorbed onto a coupled C18 cartridge that had been conditioned with methanol for cleanup. After washing with ultrapure water (2 mL) and drying, the polar HAA were eluted with a 0.8 mL of methanol:ammonia (25%) mixture (90:10, v/v), dried under nitrogen, and redissolved in 100 μL of caffeine solution (internal standard). A Gynkotek HPLC-system (Gynkotek, Germering, Germany) with a M480 pump, a Gina 50 autosampler, a degasser (DG 1310 S) connected to a fluorescence detector (RF 1002), and a diode array detector (UVD 320) were used for the analytical determination of HAA. The system was equipped with a Gynkosoft chromatography data software (version 5.50). HAA were analyzed using a TSK-gel ODS-80TM 250−4.6 mm, 5 μm analytical column (Tosoh Bioscience, Stuttgart, Germany) and a Supelguard LC-18-DB guard column (Supelco, Bellefonte, PA). The injection volume was 60 μL. The mobile phase contained 10 mM triethylamine adjusted with phosphoric acid to pH 3.2 as eluent A, 10 mM triethylamine adjusted to pH 3.6 as eluent B, and acetonitrile as eluent C. HAA were separated with the following gradient program: 82−75% A, 10% B, and 8−15% C from 0 to 10 min; 0% A, 85−75% B, and 15−25% C from 10 to 20 min; 0% A, 75−55% B, and 25−45% C from 20 to 29 min; 0−82% A, 55−10% B, and 45−8% C from 29 to 33 min; 82−15% A, 10% B, and 8−75% C from 33 to 35 min. The flow rate was 1 mL/ min. The mobile phase with 75% C was run for 4 min to recover the HPLC column. The HPLC system was then reset to its original starting conditions and equilibrated for 10 min. UV-detection was carried out at 258 nm, and a 3D-field was used for spectral plots at 200−360 nm. The fluorescence detector (ex/em) was adjusted to 360/450 nm at 0 min, 300/440 nm at 14 min, 265/410 nm at 22 min, 305/390 nm at 24 min, 265/410 nm at 25.5 min, and 335/410 nm at 28 min. The peaks of HAA, norharman, and harman were identified by comparing their retention times and UV-spectra with those of the corresponding standards. Quantification was performed with an external calibration (norharman, harman) or standard addition using a single point in duplicate (MeIQx, PhIP) (two samples and two standard mixture spiked samples). The limit of detection (LOD) was 0.017−0.03 ng/g for UV and 0.002−0.015 ng/g for fluorescence detection.27 Statistical Method. All data for the concentrations of HAA, the diameter of liposomes, and the ratio of shell volume to total liposome volume were subjected to the Shapiro−Wilk test to assess whether the data was normally distributed. If the data were normally distributed,

the values would be analyzed using an analysis of variance with the procedure GLM and the Tukey test (α = 0.05 and/or α = 0.01) of SAS program version 9.3 (SAS Institute Inc., Cary, NC). If the data was not normally distributed a nonparametric test (Kruskal−Wallis test, α = 0.05) would be used to determine differences. The coefficients of determination (R2) were calculated (SAS program) for the particle diameter of liposomes, as well as for the ratio of GSE to lecithin concentration.

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RESULTS AND DISCUSSION Determination of Liposomal Stability. The diameter of each liposomal preparation, with and without extract, was measured immediately after production and during storage for four consecutive weeks. Figure 1A−C shows that the diameters of the liposomes without extract were significantly smaller than those of liposomes with GSE. The 5% lecithin liposomes had the smallest z-average particle diameter when compared to the 1% and 2% trials. The differences in z-average diameter between the control (without extract) and liposomes with

Figure 1. Evaluation of z-average particle diameter of liposomal solutions with different lecithin (1−5%) and GSE (0−0.2%) concentrations, during storage (0−28 days) (n = 8). (A) 1% lecithin, (B) 2% lecithin, (C) 5% lecithin. C

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extract suggest that the polyphenolic compounds had been incorporated into the liposomal bilayer, because liposomes with extract were larger than liposomes without it (3-, 2.5-, and 1.6fold for the 1%, 2%, and 5% trials with 0.2% GSE, respectively). Particle diameters remained virtually unchanged during the four weeks of storage, an indication of the good stability of the liposomal preparations (Figure 1A−C). The ratio of GSE content to lecithin had a linear, positive correlation (R2 = 0.97, p < 0.001) to the particle diameter (Figure 2). The largest particle diameter had a ratio of 20%

Figure 2. z-Average particle diameter as a function of the ratio of content of GSE to lecithin concentration (0−20%) (n = 8).

GSE in the liposomal lecithin at 1% liposomes containing 0.2% GSE. The correlation shown in Figure 2 confirms that higher ratios of GSE led to liposomes having higher z-average particle diameters. It was not possible to encapsulate more than 20% of GSE because at higher concentrations, large GSE−lecithin aggregates were formed that rapidly formed a sediment layer at the bottom of the sample container. Such sediment-containing solutions could not be processed with the microfluidizer, i.e., the aggregates blocked the homogenization valve. In Figure 3, the zeta potential of liposomes containing GSE concentrations ranging from 0% to 0.2% is depicted as a function of storage time. The zeta potential decreased on average about −14 mV for 0.1% GSE and −9 mV for 0.2% GSE. Similar results had been reported in an earlier study, where liposomes containing GSE had a more negative surface charge with respect to the controls.21 The zeta potential slightly decreased in all liposomal systems during storage (Figure 3). Determination of Total Phenolic Compounds by Folin−Ciocalteu Assay. The analysis of the polyphenolic content using the Folin−Ciocalteu assay confirmed that most of the polyphenolic compounds had been incorporated in liposomes (Figure 4). The mean encapsulation efficiency for all liposomal systems containing GSE was 94% ± 14.9%. For example, the amount of unbound polyphenols was particularly low in liposomes containing 5% lecithin, suggesting that the ratio of GSE to lecithin affected the amount being encapsulated (Table 2). The fact that higher ratios of GSE to lecithin resulted in higher amounts of phenols being present in liposomes is in agreement with results from a prior study.21 To gain more insight into the distribution of polyphenols between the liposomal membrane and the interior, the volume of the inner compartment of liposomes and thus the concentration of GSE there can be calculated by considering the ratio of shell to total volume of liposomes Vshell/Vtotal (eq 2) (Table 2).

Figure 3. Zeta potential of the liposomes containing GSE as a function of storage time (0−28 days) (n = 8). (A) liposomes 2% containing 0− 0.2% GSE, (B) liposomes 5% containing 0−0.2% GSE.

Figure 4. Concentration of phenolic compounds (calculated as gallic acid equivalents) in liposomal solution, as a function of the ratio of GSE to lecithin content (0−20%) (n = 4).

Vshell = Vtotal D

4 πr 3 3 2

4

− 3 π (r2 − Δr )3 4 πr 3 3 2

(1)

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Table 2. Theoretical Calculations of the Ratio of Shell to Total Volume of Liposomes (Vshell/Vtotal)a concentration of lecithin (% w/w) 1%

2%

5%

concn of GSE (%w/ v)

particle radius (nm)b−d

Vshell/Vtotal (% v/v)b−d

particle radius (nm)b−d

Vshell/Vtotal (% v/v)b−d

particle radius (nm)b−d

Vshell/Vtotal (% v/v)b−d

0 0.1 0.2

16.5 ± 0.23Aa 55.7 ± 1.80Ab 64.0 ± 2.18Ac

56.1 ± 0.6Aa 20.0 ± 0.1Ab 17.6. ± 0.1Ac

14.1 ± 0.42Ba 33.4 ± 0.2Bb 42.3 ± 0.1Bc

63.2 ± 1.3Ba 31.8 ± 0.2Bb 25.8 ± 0.1Bc

19.7 ± 0.42Ca 26.1 ± 0.33Cb 29.0 ± 0.31Cc

49.3 ± 1.6Ca 39.3 ± 0.2Cb 36.0 ± 0.3Cc

Calculations were carried out with eq 2, considering Δrshell = 4 nm and the measured radius of each liposome. bData are reported as mean ± SD. Means with different letters are significantly different (p < 0.05). dLower case for 0−0.2% GSE, upper case for 1−5% lecithin.

a c

Table 3. Concentrations of HAA in Fried Beef Patties after Marination with GSE, Rapeseed Oil, and Liposomes (1, 2, and 5% w/w lecithin) Containing GSE (0.1% and 0.2%) (n = 6, p < 0.01) HAA in fried beef pattiesa samples

MeIQx (ng/g) mean ± SD

control I (rapeseed oil) control II 0.1% GSE control III 0.2% GSE lip. 1% lec. lip. 1% lec., 0.1% GSE lip. 1% lec., 0.2% GSE lip. 2% lec. lip. 2% lec., 0.1% GSE lip. 2% lec., 0.2% GSE lip.5% lec. lip. 5% lec., 0.1% GSE lip. 5% lec. 0.2% GSE a

1.9 1.2 1.3 1.2 1.0 1.1 1.0 0.9 1.3 1.4 1.2 1.4

± ± ± ± ± ± ± ± ± ± ± ±

0.06a 0.07b 0.27b 0.09b 0.13b 0.10b 0.05b 0.18b 0.01b 0.14b 0.17b 0.39b

PhIP (ng/g) mean ± SD 1.0 0.5 0.8 1.2 0.5 0.9 0.7 1.1 1.2 1.9 0.3 1.4

± ± ± ± ± ± ± ± ± ± ± ±

0.21bcd 0.12cd 0.21bcd 0.04bc 0.15cd 0.11bcd 0.24bc 0.17bc 0.10abc 0.69a 0.03d 0.16ab

norharman (ng/g) mean ± SD 9.2 8.9 11.3 9.4 9.2 9.0 10.4 10.5 11.9 9.9 4.1 11.0

± ± ± ± ± ± ± ± ± ± ± ±

0.64cd 0.6d 0.27ab 0.05cd 0.64cd 0.45cd 0.26cd 1.03abc 0.93a 0.02bcd 0.01e 0.01ab

harman (ng/g) mean ± SD 3.8 3.3 4.0 4.6 4.7 4.6 4.4 4.8 5.7 4.4 2.0 4.7

± ± ± ± ± ± ± ± ± ± ± ±

0.26cd 0.11d 0.2bcd 0.02bc 0.24bc 0.78bc 0.12bc 0.56ab 0.36a 0.01bc 0.01e 0.01bc

lip. = liposomes, lec. = lecithin, and GSE = grape seed extract. Means with different letters are significantly different.

Vshell (r − Δr )3 =1− 2 3 Vtotal r2

norharman and harman were found in the nonpolar fraction (data not shown). The average recoveries for HAA were 61% for MeIQx, 47% for PhIP, and 55% for norharman and harman, which confirms the high efficiency of the method used for such substances. These recoveries are similar to those reported in other studies.27 Table 3 shows the content of HAA in fried beef patties. The most predominant HAA found in the beef samples for the polar fractions was MeIQx. Generally, the content of polyphenols had an inhibiting effect on the formation of MeIQx during frying of beef. In comparison to control samples (marinated with rapeseed oil), a significant decrease of MeIQx (p < 0.01) was observed with respect to all other samples. However, no significant differences were found between different experimental treatments marinated with liposomes and liposomalencapsulated GSE (Table 3). In contrast, significant differences were detected in the MeIQx concentration in the control trial, in particular between the samples marinated with rapeseed oil and those marinated with pure GSE. The concentration of extract (0.1% and 0.2%) did not have an impact on the formation of MeIQx. However, significant differences were detected between the control treatment (marinated with rapeseed oil), and the encapsulation trials (1%, 2%, and 5% of soy lecithin) regardless of the soy lecithin percentage. The MeIOx concentration was approximately 1.6-fold higher in the sample marinated with rapeseed oil than in samples marinated with liposomal-encapsulated GSE solution. Various studies have demonstrated the positive effect exerted by molecules with antioxidant activity on the inhibition of HAA formation, with a particular focus on the inhibition of formation of MeIQx.30,31 The marination of meats with barbeque sauces, mixtures of spices, red wine, beer, or plant extracts before frying

(2)

with r2 being the mean radius after microfluidization. A shell thickness of 44.5 Å ± 0.3 Å for unilamellar 1,2-dimyristoylphosphatidylcholine vesicles of approximately 4 nm (Δr) has been reported for small unilamillar vesicular liposomes containing a single bilayer.28,29 The ratio of the core volume to the total volume thus increased from 37% to 82% in liposomes containing extract (Table 2). Consequently, at higher loading capacities of GSE where lipsomes have larger particle diameters, GSE is increasingly located in the interior of liposomes rather than in the membrane, simply due to spatial constraints (Figure 2). Effect on the Formation of HAA in Fried Beef Patties. The effectiveness of application of liposomal-encapsulated GSE on beef patties on weight loss and formation of HAA was assessed. The weight loss of patties during frying was determined as the weight difference of the beef patties before and after frying. The internal temperature was continually monitored by using a data logger, and it was around 72 °C at the end of the frying process. The mean weight loss of the patties was between 29.3% and 34.9% for the different batches, with an overall standard deviation of 2.7%. The results of the weight loss measurements showed that all patties were subjected to similar frying conditions. In the polar fraction of HAA method, the two most important polar HAA (MeIQx and PhIP) were identified. The HPLC-chromatograms are shown in Figure S1 in Supporting Information. The concentrations of PhIP were quantified using the higher sensitively fluorescence detection. β-Carbolines E

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Figure 5. Schematic overview of HAA formation (e.g., MeIQx) and the proposed mechanism with possible interactions between the marinade consisting of liposome-entrapped GSE and the meat matrix.

mostly led to a reduction in the HAA content.5−7,25,32−34 Similarly in the present study, an inhibitory effect on formation of HAA was found when compounds with antioxidant activities were used to marinate the beef patties. However, liposomal encapsulation of GSE did not lead to a significant improvement in the inhibition of the HAA formation compared to the use of free GSE. Complex interplays take place that depend on oil/ lipid type and concentration. For example, authors reported an increasing effect of oxidized oil on the formation of HAA after different storage times.35,36 These somewhat disappointing results with respect to MeIQx may be due to the formation mechanism of polar amines, which are generated by the reaction of hexose and amino acids via Maillard and Strecker degradation reactions, whereby the free radical intermediate form of pyridines and pyrazines will react with creatine.37,38 The apparent positive impact on the diminution of polar MeIQx may not be solely due to the action of the polyphenols but may be attributed to an antioxidant effect exerted by soy lecithin, which contained 0.18% DL-α-tocopherols, according to its specification. Therefore, approximately 4−8 mg of polyphenolic compounds and 0.2−0.9 mg of α-tocopherols were calculated per patty marinated with GSE-entrapped liposomes. With PhIP, a different behavior was observed compared to that of the other polar HAA. No differences at a level of significance of p = 0.01 were found between the control and samples treated with GSE, regardless of the polyphenol concentration. In other words, an antioxidant effect from the polyphenolic compounds was not evident. A significant decrease of PhIP was detected in the trials with liposomes prepared with 5% of soy lecithin that contained 0.1% GSE (p < 0.05). Nevertheless, no systematic correlation between the concentration of PhIP and the GSE content in liposomes could be established. A significant increase of norharman compared to the rapeseed oil control was observed after marination with 0.2% pure extract, and 2% and 5% liposomes containing 0.2% GSE. The β-carboline norharman levels varied between 8.9 and 11.9 ng/g, except for the 5% liposomes containing 0.1% GSE (Table 3). Harman concentration ranged from 3.3 to 5.7 ng/g. The β-

carboline harman generally increased with GSE content with results being significantly different for samples marinated with 2% liposomes containing GSE. These results are similar to those reported in an earlier study, where β-carbolines increased with the addition of GSE.23 The concentration of norharman and harman are comparable to those reported by Totsuka et al.39 One can conclude from these results that the use of GSE is somewhat detrimental to the formation of β-carbolines norharman and harman and that the application form (encapsulated or free) does not affect the β-carboline levels. This may be related to the formation mechanism of norharman and harman, which does not involve a free radical action and, for this reason, cannot be inhibited by the addition of antioxidants such as polyphenols.37 In the formation pathway of these compounds, tryptophan has been identified as their precursor, whereas glucose enhances norharman formation. In aqueous model systems containing creatine, glucose, and various single amino acids, norharman and harman were formed in mixtures containing isoleucine, arginine, phenylalanine, and tryptophan, and this mixture combined with tyrosine generated norharman upon heating at 180 °C for 10 min. 40 When iron (Fe2+) and copper (Cu2+) were added to a model system, norharman was generated even at temperatures as low as 40 °C.40 Mechanistic Insights. The fact that entrapped GSE polyphenols in liposomes had no additional inhibitory effect on the formation of MeIQx and PhIP is somewhat surprising, considering that liposomal-encapsulated polyphenols have previously been found to improve inhibition of, for example, lipid oxidation in model systems.21 Moreover, in another study, the use of a different encapsulation system, a water-in-oil marinade with GSE being encapsulated in water droplets suspended in an oil phase, was found to significantly reduce formation of the mutagenic compounds MeIQx and PhIP compared to water-in-oil marinade without extract.23 There, the water-in-oil marinade contained 0.2−0.8% GSE (pH 5.6) and had been applied before frying of beef patties. In consequence, the content of MeIQx and PhIP decreased by approximately 57% and 90%, respectively.23 F

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ingredient such as for example the polyphenol, but it must be designed such that is ensures that high concentrations of an active compound are present at the required site of release. If the reaction takes place at the surface of a product, then the generation of small particles that are compatible with the properties of the food matrix may not be ideal. Rather, larger particles with a certain “incompatibility”, preventing a migration into the matrix, should be manufactured. In contrast, if the reaction takes place in the interior of the matrix, small carriers that are compatible with the matrix may be well suited to influence the reaction. This study therefore should be helpful to food manufacturers to better understand chemical or physical interactions in complex food matrixes (such as meat), by means of encapsulated active compounds as well as the encapsulating material.

The observed differences between the results in this study and the results of the above cited former studies may be due to a variety of physical mechanisms that are influenced by the structure and physical properties of the encapsulation system used and its interaction with the application matrix (Figure 5): (i) Diffusion and partitioning behavior: Liposomes containing GSE had a very small particle size and a hydrophilic surface and thus were water-soluble. Thus similar to free GSE, once applied on the surface of the meat matrix, they are able to rapidly diffuse into the interior of the meat matrix. Once in the interior, they are effectively unable to inhibit the formation reaction of HAA that occurs mostly at the surface where high temperatures are prevalent. This is notably different from the above-described water-in-oil system, which is hydrophobic and has much larger particle sizes. There, water droplets containing GSE are enclosed by an oil matrix and are thus mostly present at the surface of the meat product. The heating may lead to an evaporation of the internal water droplets, which in turn leads to a release of the polyphenols able to inhibit the reaction. (ii) Interaction behavior: The encapsulation of GSE in liposomes may have also not prevented the polyphenols from interacting with dissolved proteins in the meat matrix. During grinding, some of the proteins are solubilized, leading to a characteristic binding of meat and fat particles in ground beef patties. Studies have shown that the interaction of both phosphatidylcholine liposomes and polyphenols with dissolved proteins generally leads to the formation of protein− polyphenol41 and liposomes−protein complexes,42 respectively, rendering polyphenols unable to effectively inhibit radical reactions. These interactions may be driven by hydrophobic as well as electrostatic interactions, the latter being due to the fact that both the liposomes and the free GSE were negatively charged (Figure 3) while the surface charge of meat proteins at the pH of the buffered marinade of 3.8 was below the isoelectric point and therefore positively charged. The former may be due to the fact that most proteins possess significant surface hydrophobicities, and because GSE was bound mostly to the surface or was part of the liposomal membrane, an interaction may readily occur. This would again be noticeably different from the functionality of a water-in-oil emulsion, in which the polyphenols are protected from interaction with proteins by the oil phase that surrounds the internal water droplets containing the GSE. Finally, the formation of PhIP differs from the formation of MeIQx. On the basis of literature studies, while glucose does not seem to be a necessary precursor when using dry heating in a model system, it may however enhance or inhibit the formation of PhIP.43 In a model system with only phenylalanine and creatinine as precursors, the Strecker aldehyde phenylacetaldehyde is initially formed. In a second step an aldol condensation of the aldehyde with creatinine and subsequent dehydration takes place. The products of the addition as well as the condensation product were identified in the model system and in heated meat.44 Our working hypothesis that a liposomal encapsulation may inhibit HAA formation due to the increased surface of the liposomes was therefore not supported by the data obtained. In fact, the system proved to be ineffective with results being analogous to those of trials with free GSE. However, the study led to new mechanistic insights that should help to more effectively and rationally design encapsulation systems for antioxidants. A functional encapsulation system for polyphenols must not only be able to contain high contents of the active

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ASSOCIATED CONTENT

* Supporting Information S

Figure S1: HPLC UV (258 nm) and fluorescence chromatograms of polar HAA in fried beef patties marinated with 5% liposomes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 711 459 22293. Fax: +49 711 459 24446. E-mail: [email protected]. Funding

This project was supported financially by funds from the University of Hohenheim Experiment Station. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Lipoid GmbH and the Plantextrakt−Martin Bauer Group for donation of sample materials.

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ABBREVIATIONS USED GSE, grape seed extract; Lip, liposomes; Lec, lecithin; SD, standard deviation; HAA, heterocyclic aromatic amines; IQ, 2amino-3-methylimidazo[4,5-f ]quinoline (CAS no.: 76180-966); IQx, 2-amino-3-methylimidazo[4,5-f ]quinoxaline (CAS no.: 108354-47-8); MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f ]quinoline (CAS no.: 77094-11-2); MeIQx, 2-amino-3,8dimethylimidazo[4,5-f ]quinoxaline (CAS no.: 77500-04-0); 4,8-DiMeIQx, 2-amino-3,4,8-trimethylimidazo[4,5-f ]quinoxaline (CAS no.: 95896-78-9); 7,8-DiMeIQx, 2-amino3,7,8-trimethylimidazo[4,5-f ]quinoxaline (CAS no.: 92180-795); PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (CAS no.: 105650-23-5); Trp-P-1, 3-amino-1,4-dimethyl-5Hpyrido[4,3-b]indole (CAS no.: 62450-06-0), monoacetate (CAS no.: 68808-54-8); Trp-P-2, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (CAS no.: 62450-07-1); Glu-P-1, 2-amino-6methyldipyrido[1,2-a:3′,2′-d]imidazole (CAS no.: 67730-11-4); Glu-P-2, 2-aminodipyrido[1,2-a:3′,2′-d]imidazole (CAS no.: 67730-10-3); AαC, 2-amino-9H-pyrido[2,3-b]indole (CAS no.: 26148-68-5); MeAαC, 2-amino-3-methyl-9H-pyrido[2,3b]indole (CAS no.: 68006-83-7); harman, 1-methyl-9H-pyrido[3,4-b]indole (CAS no.: 486-84-0); norharman, 9H-pyrido[3,4b]indole (CAS no.: 244-63-3) G

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